A METHOD, AN APPARATUS AND A KIT FOR RAPID SCREENING OF FUNGAL ORGANISMS

FIELD OF THE INVENTION

The present invention provides a method for identifying the presence of Candida spp., in particular Candida albicans in a given sample. The invention also provides an apparatus and a kit for identifying the presence of Candida spp., in particular Candida albicans in a given sample.

BACKGROUND OF THE INVENTION

Invasive fungal infections are being increasingly recognized as a major threat among the immunosuppressed and immunocompromised population of patients. Candida and Aspergillus species are the most common causes of fungal infections, but other yeasts and filamentous fungi are emerging pathogens. C. albicans, is the most significant pathogenic species and is associated with significant morbidity and mortality.

Although infections caused by Candida and Aspergillus spp. continue to remain a threat, other fungal pathogens are gaining prominence; especially among the high risk population such as stem cell and solid organ transplant patients, cancer patients, neonates, HIV patients and surgical patients. The rapid emergence of new treatment options offering substantial advantages over the past two decades has facilitated much broader use of antifungals in empiric and prophylactic settings. On the contrary the introduction of key therapeutics to tackle the common pathogen C. albicans has led to the increase in the burden due to other fungal pathogens such as aspergillus. As products used to treat this latter group become more widely used, another shift in epidemiology may occur, contributing to the rise of further pathogens (such as Fusarium and Scedosporium species) for which there are few treatment options. Owing to the mortality and morbidity due to various fungal pathogens, especially C. albicans, a specific and rapid diagnosis of fungal pathogens is needed in the art.

Candida albicans is a fungus responsible for various forms of candidiasis, a condition which may be found in normal and immunocompromised patients. C. albicans infects both skin and mucous membranes, and is also capable of causing life-threatening infections. A number of putative virulence factors have been proposed and the ability of the organism to convert morphologically from blastoconidia to filamentaneous forms has been a subject of intensive study. The conversion to the hypha is accompanied by expression of novel antigens on the filamentaneous form. The diagnosis of Candida species is crucial in treatment of infections by fungal species.

Although C. albicans remains the most prevalent Candida species, infections caused by other Candida species such as C. glabrata, C. dubliniensis, C. guilliermondii, C. parapsilosis, C. tropicalis and C. kefyr, have increased in relative prevalence in the past few years. Several attempts have been made to generate reagents which can specifically differentiate C. albicans from these species. C. dubiliniensis, associated with oral infections in immunocompromised individuals, like C. albicans is unique in forming germ tubes, the precursor to the true hypha. Owing to the phenotypic and genotypic similarity between the two species, particularly the ability to form filaments, differentiating C. albicans and C. dubiliniensis has been challenging. Further owing to the other fungal organisms gaining a pathogenic status, there is need in the art to be able to specifically identify these pathogens.

The current state-of-the-art test for diagnosing the infective organism in case of fungal pathogens is based on growth characteristics of the organism in specialized differential media along with the well known germ tube formation test as well as the antibody based agglutination test. While the diagnosis based on growth takes several hours to days to yield convincing result, the germ tube formation test can give false positive results. The false positive or false negative test results can affect seriously the treatment regimen and in turn the outcome of the treatment.

The anti-Candida antibody based agglutination test although rapid and quantitative, lacks specificity and the ability to differentiate between various species of Candida. Moreover, many immunocompromised groups of patients have a low serum antibody titer which may attribute to false positive results.

Molecular techniques like PCR, DNA fingerprinting and pulsed gel electrophoresis are known in the field but these are time consuming and cannot be applied to large number of isolates (Sullivan D, 1998). An approach of differentiating C. albicans and C. dubliniensis based on fluorescent in situ hybridization with peptide nucleic acid probes (PNA-FISH) (Odds FC, 1998) has been reported. However this is more labor intensive and expensive. Bliss et al (2003) have disclosed the use of phage display library to identify single chain antibody fragments that specifically recognize the filamentous form of C. albicans. Imaging techniques are known in the art for fungal detection. This technique though powerful, uses ionizing radiation, which are harmful. Conventional techniques like histology and cytology based detection of fungal pathogens are unable to specifically differentiate various fungal pathogens in the immunocompromised patients. Furthermore, the difficulty in isolating sample from these patients and the occurrence of false positive results make this technique unreliable for the detection of fungal pathogens.

Owing to the rising prevalence of fungal, especially Candida infections, there is a need for improved method(s) and means for the detection of specific Candida species in various samples with specificity. As such, a method that solves or at least alleviates the problems and limitations of the prior art will be preferable.

SUMMARY OF THE INVENTION

In its one of the general aspects, the invention disclosed herein provides an efficient, simple, rapid and cost effective method for the detection of Candida spp. in given samples. In particular the method is for the detection of Candida albicans.

Accordingly, in one aspect, the invention provides a method for the detection of Candida spp. in given samples, comprising the steps of: incubating the test sample with at least one peptide selected from SEQ ID NO. 1, homolog or a fragment thereof;

detecting the binding of the peptide to the sample in at least one measurable binding assay;

quantitating the intensity of binding of the peptide to the sample; and comparing the binding and/or intensity with at least one control sample.

In still another aspect, the invention provides a method for the detection of Candida spp. in given samples, comprising the steps of: incubating the test sample with at least one an amino acid sequence making up a peptide selected from SEQ ID NO: 1, homologue or a fragment thereof; detecting the binding of the peptide to the sample in at least one measurable binding assay;

quantitating the intensity of binding of the peptide to the sample; and comparing the binding and/or intensity with at least one control sample.

In still another aspect, the invention provides a method for the detection of Candida spp. in given samples, comprising the steps of: incubating the test sample with host cells expressing at least one peptide selected from SEQ ID NO: 1 or a homologue or a fragment thereof; detecting the binding of the peptide to the sample in at least one measurable binding assay;

quantitating the intensity of binding of the peptide to the sample; and comparing the binding and/or intensity with at least one control sample.

In still further aspect, the invention provides a method for the detection of Candida spp. in given samples, comprising the steps of: incubating the test sample with host cells capable of binding to at least one Candida species;

detecting the binding of the host cells to the sample in at least one measurable binding assay;

quantitating the intensity of binding of the peptide to the sample; and comparing the binding and/or intensity with at least one control sample.

In its other general aspect, the invention disclosed herein provides an apparatus for the detection of Candida spp., comprising an inert means, with at least one chamber for holding a test sample and optionally at least one control chamber wherein both chambers include plurality of cells stuck on their surface.

In its still one more general aspect, the invention disclosed herein provides a kit for the detection of Candida spp., in given samples comprising an apparatus for detection of Candida spp.; reagent 1 including phage clone or peptide having SEQ ID No. 1 or a homologue or a fragment thereof conjugated with an enzyme; and reagent 2 including the substrate for the enzyme of reagent 1.

BRIEF DESCRIPTION OF THE DRAWINGS

So that the manner in which the above recited features of the present invention can be understood in detail, a more particular description of the invention, briefly summarized above, may be had by reference to various embodiments, some of which are illustrated in the appended drawings. It is to be noted, however, that the appended drawings illustrate only typical embodiments of this invention and are therefore not to be considered limiting of its scope, for the invention may admit to other equally effective embodiments. FIG 1 represents Phage library technique used in this study.

FIG 2 shows the plaque assay with clones derived based on phage library that showed binding to various Candida spp.

FIG 3a and 3b shows clones derived based on phage library showing specific binding ability to Candida albicans in an ELISA assay.

FIG 4 shows the binding of Candida to clones derived based on phage library in an immuno-fluorescence assay

FIG 5 shows the binding of the fluorescent tag, detecting the presence of a clone derived based on phage library on the periphery of the cells.

FIG 6a and 6b is a representative example of an apparatus for the detection of Candida spp.

BRIEF DESCRIPTION OF THE SEQUENCES

SEQ ID NO: 1 refers to a generic 12mer peptide the derivatives of which are the isolated epitopes in the phage display library capable of binding to Candida spp.

SEQ ID NO: 1:

[E/S/A/T]XX[A/S/T/P/H]X[ILPMA][IVMHPS][PSTDQN][STAPFL][SATRYL]X, where X is any amino acid.

SEQ ID NO: 11 to 21 refers to nucleotide sequences encoding the 12mer peptides of the invention having SEQ ID NO: 1

Where W=A/T
Y=T/C
R=A/G
S=C/G
N= A/T/G/C
M=A/C

DETAILED DESCRIPTION OF INVENTION

Unless otherwise explained, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. It is further to be understood that all base pair sizes or amino acid sizes, and all molecular weight or molecular mass values, given for nucleic acids or polypeptides are approximate, and are provided for illustration. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of this disclosure, suitable methods and materials are described.

Bibliographic references mentioned in the present specification are for convenience listed in the form of a list of references and added at the end of the examples. The whole content of such bibliographic references is herein incorporated by reference.

"Sensitivity" and "specificity" as used herein are statistical measures of the performance of a binary classification test. "Sensitivity" measures the proportion of actual positives which are correctly identified as such, for example the percentage samples identified as harboring the C. albicans pathogen. "Specificity" measures the proportion of negatives which are correctly identified, for example the percentage of control samples identified as not having C. albicans. A theoretical, optimal prediction can achieve 100% sensitivity, i.e. all samples isolated from candidiasis patients should be identified as harboring C. albicans in them. A100% specificity would mean that none of the control samples was positive for Candida albicans.

"Detection", "Detecting" or "identifying" as used herein refers to determining the nature or the identity of a condition (say of a disease like candidiasis). Detection may be accompanied by a determination as to the severity of the condition. "Quantitating" as used herein refers to determining the number of organisms in the given test sample that is attributing to the condition, for example candidiasis.

"peptides" refers to a molecular chain of amino acids and does not refer to a specific length of the product. These peptides may further make polypeptide, oligopeptides and proteins and the same are included within the scope of the invention based on the peptides disclosed herein. This term is also intended to include peptides that have been subjected to post-expression modifications such as, for example, glycosylations, acetylations, phosphorylations, and so forth.

"Protein" refers to any molecular chain of amino acids that is capable of interacting structurally, enzymatically or otherwise with other proteins, polypeptides or any other organic or inorganic molecule.

Fragment" refers to an amino acid sequence of a peptide or protein that is shorter than the entire peptide or protein, but contains at least about 25 consecutive amino acids of the full peptide or protein.

"Epitope" refers to a part of a protein that specifically binds an antibody by fitting into the antibody-combining site.

"Test sample" refers to a biological material suspected of containing the Candida spp. The test sample may be derived from any biological source, such as a physiological fluid, including, blood, interstitial fluid, saliva, ocular lens fluid, cerebral spinal fluid, sweat, urine, milk, ascites fluid, mucous, nasal fluid, sputum, synovial fluid, peritoneal fluid, vaginal fluid, menses, amniotic fluid, semen, and so forth. Besides physiological fluids, other liquid samples may be used such as water, food products, and so forth, for the performance of environmental or food production assays. In addition, a solid material suspected of containing the Candida spp. may be used as the test sample. The test sample may be used directly as obtained from the biological source or following a pretreatment to modify the character of the sample. For example, such pretreatment may include preparing plasma from blood, diluting viscous fluids, and so forth. Methods of pretreatment may also involve filtration, precipitation, dilution, distillation, mixing, concentration, inactivation of interfering components, the addition of reagents, lysing, etc. Moreover, it may also be beneficial to modify a solid test sample to form a liquid medium or to release the analyte.

In general, the present invention is directed to methods, an apparatus and a kit for detecting the presence of at least one Candida spp in a given sample, in particular Candida albicans in a sample in an efficient, simple, rapid and cost effective manner.

Accordingly, in one embodiment, the present invention provides a method for detecting the presence of at least one Candida spp. in a test sample, in particular Candida albicans in a sample. The method comprises the steps of: incubating the test sample with at least one peptide with SEQ. ID NOs. 1 homolog or a fragment thereof;

detecting the binding of the peptide to the sample in at least one measurable binding assay;

quantitating the intensity of binding of the peptide to the sample; and comparing the binding and/or intensity with at least one control sample.

In another aspect, the invention provides a method for the detection of Candida spp. in given samples, comprising the steps of: incubating the test sample with at least one an amino acid sequence making up a peptide selected from SEC* ID NO: 1, homologue or a fragment thereof; detecting the binding of the peptide to the sample in at least one measurable binding assay;

quantitating the intensity of binding of the peptide to the sample; and comparing the binding and/or intensity with at least one control sample.
In still another aspect, the invention provides a method for the detection of Candida spp. in given samples, comprising the steps of: incubating the test sample with host cells expressing at least one peptide selected from SEQ ID NO: 1 or a homologue or a fragment thereof; detecting the binding of the peptide to the sample in at least one measurable binding assay;

quantitating the intensity of binding of the peptide to the sample; and comparing the binding and/or intensity with at least one control sample.

In still further aspect, the invention provides a method for the detection of Candida spp. in given samples, comprising the steps of: incubating the test sample with host cells capable of binding to at least one Candida species;

detecting the binding of the host cells to the sample in at least one measurable binding assay;

quantitating the intensity of binding of the peptide to the sample; and comparing the binding and/or intensity with at least one control sample.

Though the above embodiments are directed towards the detection of Candida spp., in particular for the specific detection of Candida albicans, the methods disclosed hereinabove can be extrapolated for the detection of other Candida species and other fungal pathogens.

The detection or quantitating of the organisms in the sample can be done by any of the techniques known in the art, for example enzyme-linked immunoassay (ELISA or EIA) or immunofluorescence but not limited to these techniques. As used herein "detecting the presence of fungal pathogen" refers to a measurable parameter of the presence of binding event between the organism in the provided sample and the epitope displayed by the host cell.

For example, presence of binding can be detected using several well-recognized binding assays, for example the ELISA described in the further sections. Cell free assays can be used to measure the binding of the peptides of the current invention by various assays including but not limited to fluorometric, assay or flow cytometric assay. The assays and means mentioned herein are exemplary and by no way limiting.

In other embodiments, the present invention provides peptides capable of binding to Candida spp., in particular to Candia albicans. Such peptides, comprises at least one amino acid sequence selected from one of the derivatives of SEQ ID NO: 1, however, the invention is not limited to these. Any other homologue, variant of these peptides may be encompassed by the invention. Further any other homologous peptides may show binding to other fungal organism and/or Candida spp. The peptides showing binding to Candida spp. known in the art, for example C. glabrata, C. dubliniensis, C. guilliermondii, C. parapsilosis, Crytococcus neoformans, Aspergillus sp. C. tropicalis and/OR C. kefyr or the like may be encompassed by the present invention.
The invention also provides a polynucleotide encoding the peptide/s of the invention, homologue or fragment thereof.

The peptides of the invention can be used as antifungal agents, specifically against C. albicans. Accordingly, the present invention provides at least one linear peptide comprising the amino acid sequence of any one of the derivatives of SEQ ID NO: 1, homolog or a fragment thereof.

The peptides of the invention can be encoded by nucleotide sequences having SEQ ID. 11 to 20.

Any of the nucleotides described above may be comprised in any suitable vector. The vector may be comprised in a suitable host cell. Accordingly the vector may in at least one host cell, wherein the host cell may be capable of expressing at least one peptide selected from the derivatives of SEQ ID NO: 1, homolog or a fragment thereof. The host cell may be capable of binding to at least one Candida spp., in particular Candida albicans. The host cell may be a phage.

Peptide/s, homologs and/or fragments thereof, capable of binding to Candida spp, in particular Candida albicans that can aid in the detection of these organisms in various samples have been derived using a phage display library.

In one embodiment, the present invention provides a method of isolating peptides capable of binding to Candida spp, in particular Candida albicans that can aid in the detection of these organisms in various samples using a phage display peptide library, the specifics of such method is described herein later under the methods of the invention.

In one more embodiment, the present invention provides and apparatus for the detection of at least one Candida spp., more particularly Candida albicans in a sample. The representative example of an apparatus on the invention is illustrated in FIG 6A and 6B. The apparatus comprises of a first chamber for holding a test sample in which the presence of Candida spp. needs to be detected. A second chamber is optionally provided for holding a control sample for validation. A plurality of such chambers maybe provided in an alternate embodiment of the invention for simultaneous detection of a plurality of fungal pathogens.

In one embodiment of the invention, the floor of the chamber is pre-coated with cells extracted from a culture of C. albicans. The pre-coated chambers can also include cells from at least one Candida species selected from the group consisting of C glabrata, C. dubliniensis, C. guilliermondii, C. parapsilosis, C. tropicalis and/OR C. kefyr.

Alternately, the chambers can also be pre-loaded with a predetermined quantity of at least one from the following:

- an amino acid sequence selected from the derivative of SEQ ID NO: 1, homologue or a fragment thereof,

- peptide having SEQ ID NO: 1, homologue or a fragment thereof,

- a host cell expressing at least one peptide having SEQ ID NO: 1 or a homologue or a fragment thereof, and a host cell may be capable of binding to at least one Candida species selected from the group consisting of C. glabrata, C. dubliniensis, C. guilliermondii, C. parapsilosis, C. tropicalis and/or C. kefyr. The Candida species may be Candida albicans.

The "control" for example, may not be harboring any of the organisms being tested for. The control may exhibit decreased binding intensity in a direct binding assay and display high binding intensity in an indirect competitive assay. Accordingly, the presence of a higher binding intensity in the direct binding assay and the decrease in binding intensity in an indirect competitive assay compared to the negative control may indicate the presence of the said organism in the test sample. The "control value" may also be an average value in expression obtained from a selected population.

In one more embodiment, the present invention provides a kit for the detection of Candida spp. more particularly Candida albicans in given samples comprising an apparatus for detection of Candida spp.; reagent 1 including phage clone conjugated with an enzyme; and reagent 2 including the substrate for the enzyme of reagent 1. In alternate embodiments, reagent 1, in place of phage clone may comprise of peptides selected from the derivative of SEQ ID NO: 1, homolog or a fragment thereof. Enzyme that may be used as part of reagent 1 may be selected from any suitable enzyme, for example but not limiting to Horse radish peroxidase, alkaline phosphatase and beta-D-galactosidase. The substrate that may be used as a part of reagent 2 may be selected based on the enzyme used in reagent 1. The substrate that may be included in reagent 2 can be selected from but not limiting to TMB (3.3',5,5'-tetramethylbenzidine), (pNPP (p-nitrophenyl phosphate, disodium salt) and O-nitro phenyl- b- D galactoside.

In another embodiment of the invention, an antibody is provided, wherein the antibody specifically binds to a peptide comprising of at least one amino acid sequence selected from the derivative of SEQ ID NO: 1, a homologue or a fragment thereof. The antibody may be a monoclonal, polyclonal, chimeric, humanised, single chain, Fab, Fab', F(ab)' fragments and/or F(v) portions of the whole antibody. Further, the antibodies raised can be specific to C. albicans in one example of the invention and in general to other Candida spp. in other examples of the invention.

In an alternate embodiment of the invention, an antibody is provided, wherein the antibody binds to a peptide, a homologue and/or a fragment thereof isolated for a specific fungal pathogen. The antibody may be a monoclonal, polyclonal, chimeric, humanised, single chain, Fab, Fab', F(ab)' fragments and/or F(v) portions of the whole antibody.

According to a further embodiment of the invention, there is provided a pharmaceutical composition comprising at least one peptide selected from derivative of SEQ ID NO: 1, a homologue or a fragment thereof optionally in the presence of at least one pharmaceutically acceptable excipient, diluent, carrier and/or a combination thereof.

Further, in yet another embodiment of the invention the purified antifungal peptides may be used in the presence of a pharmaceutically acceptable carrier as an antifungal agent. The peptides according to any aspect of the invention may be included in food preparations, pharmaceutical preparations, medicinal and pharmaceutical products, cosmetic products, hygienic products, cleaning products and cleaning agents, as well as any material to which the peptides could be sprayed on or adhered to wherein the inhibition of fungal growth on such a material is desired.

The method of the present invention is rapid and less laborious technique for the detection of fungal pathogens, in particular Candida spp. and still more specifically C. albicans. The methods of the present invention help to decrease in the number of false positive and false negative results which will benefit the medical community in selecting the right treatment regimen. The method is more specific for detection of fungal pathogens. An apparatus and a kit incorporating the same for detection of the fungal pathogens provides ease of performance by any individual without having a need to be performed by a trained lab technician at any place outside sterilized conditions.

Having now generally described the invention, the same will be more readily understood through further specific method of the invention and examples.

METHOD OF THE INVENTION & EXAMPLES

Inventors of the present invention have obtained peptides capable of binding to Candida spp., in particular Candida albicans that can aid in the detection of these organisms in various samples using a phage display peptide library, the specifics of such method is described herein below with the help of specific methodology embodied in following examples.

EXAMPLE 1

Isolation of phage clones using phage display library:

Process for elution of phages capable of binding to C. albicans displaying the peptides sequences having SEQ ID NO: 1 was followed as illustrated in FIG.l. However, any other process known in the art can be followed to achieve the elution of the phages.

In this example, M13 phage, displaying 12 mer random peptides fused to pill coat protein was used. Biopanning and amplification of the phages was performed according to the procedure known in the art. In brief, C. albicans hyphal cells are blocked with 2% BSA for about 2 hours at room temperature and later cells are washed with Tris buffer (TBS pH 7.3) in 1.5 mL eppendorff tubes. Phage library with a diversity of 2xlOn plaque forming units (pfu)/mL is suspended in blocking buffer (TBS pH7.3, 2%BSA) and co-incubated with the hyphal cells at room temperature for 2 hours with vigourous shaking. Unbound phages were washed with TBST buffer (TBS pH7.3, 0.1% Tween 20) elution of bound phages was performed with 80 u.1 of 0.1M glycine (pH2) for 10 min and neutralized with 20 u.1 of 1M Tris buffer. Eluted phages were amplified to get 2xl013 pfu. After first round of biopanning, amplified phage elute (2xlOn pfu/mL) from the first round was used as input for second round of biopanning. Second round was performed in the same way as with the first round except that 0.3% Tween was used for TBST washing of unbound phages. For third round of biopanning, amplified phage elute (2xl0n pfu/mL) from the second round was used as input and for washing of unbound phages 0.5% Tween was used. Third round of elute were used to infect E. coli (ER2738) cells and plated on Luria- Bertani (LB) plates containing tetracycline (40 mg/liter) to select for the growth of cells containing phage.

Phage clones that showed binding to various Candida spp. based on the plaque assay as shown in FIG 2 were used for sequencing.

Plaque assay: Individual phage clones were further tested for affinity binding by plaque assay. From overnight saturated culture, 5xlOe yeast cells and 5xl06 cells induced for hyphae were interacted with single phage clone having 2xl09 pfu/mL phages in a centrifuge tube and incubated at room temperature for 1 hour. Unbound phages were washed with TBST buffer (0.5% Tween 20 in TBS) for three times followed by two washes of TBS to remove the traces of Tween. Bound phages were eluted with 80 ul of 0.1M glycine (pH2) for 10 min and neutralized with 20 u.1 of 1M Tris buffer. The eluted phages were serially diluted and plated on LBTet plates to detect the titer values. M13 phage without displaying any peptide was used as negative control. All 50 individual phage clones were tested against yeast and hyphal cells of C. albicans for binding specificity.

The phage clones identified to interact with Candida albicans cells in the plaque assay were sequenced and the peptide sequence determined. Several clones showed identical peptide sequence.

DNA sequencing: The affinity selected phage peptides was assessed by sequencing the region of the phage genome (N-terminal of g-lll region) that coded for the inserted 12-mer peptide and translating to the amino acid sequence. Phage clones were characterized by plaque assay for specific binding, positive clones were selected, and single-stranded DNA was isolated from the recombinant phage particles of each clone. FIG 4 shows the binding of Candida to phage clone in an immuno-fluorescence assay. FIG 5 shows the binding of the fluorescent tag, detecting the presence of a clone on the periphery of the cells. The DNA was sequenced using 96g-lll reverse primer. Peptide sequence was deduced manually by translating the DNA sequence.

Phage ELISA: Phage clones selected by panning were tested by ELISA for their ability to bind specifically to C. albicans. All ELISA were performed in 96 well microtiter plates. The wells were coated with 5xl06 yeast cells of all Candida species at 37°C for 2 hours. Wells were further blocked with 2% BSA for 2 hours at 37°C. Cells were interacted with 2xl09 plaque forming units of individual phage clones in 100 u.1 of TBS for 1 hour at room temperature on orbital shaker. Cells were washed once with 0.5% TBST and incubated with 100 u.1 of mouse antiM13 primary antibody at room temperature on orbital shaker. After 1 hour of incubation with primary antibody, cells were washed once with 0.1% TBST and incubated with 100 u.1 of anti-mouse rabbit antibody conjugated with horseradish peroxidase (HRP) at room temperature for 1 hour. After secondary treatment, cells were washed once with 0.1% TBST and developed with O- phenylene diamine (OPD) as substrate. The staining reaction was stopped by adding 50 u.1 of 1M sulphuric acid. Absorbance at 490 nm was measure by using an ELISA plate reader. Anti Candida ELISA was carried out on cells of different species used above in the microtitre plates except that the phage clones were replaced with the anti-candida rabbit antibody conjugated with HRP was used directly and the enzyme activity detected as above with the OPD substrate. FIG 3a and 3b shows phage clones showing specific binding ability to Candida albicans in an ELISA assay.

EXAMPLE 2

Detection of Candida albicans in a sample assay using an apparatus and kit of the invention:

The test sample was collected using a swab over suspected mucosal surface or other samples containing yeast form of Candida cells. The sample was applied to the inert stick coated with cells on two spots. The sample was applied only on one of the spots. Reagent 1 containing the phage conjugated with suitable enzyme for example horseradish peroxidase (HRP) was added and allowed to interact for 10 minutes. The stick was then washed under tap water gently and reagent 2, which contained the chromogenic substrate for example 3.3',5,5'-tetramethylbenzidine was added. Color developed on both sides. The reduced color intensity indicated the presence of Candia albicans in the test sample.

REFERENCES:

1. Bliss JM, Sullivan MA, Malone J, Haidaris CG: Differentiation of Candida albicans and human antibody single-chain variable fragments specific for hyphae; Journal of Clinical Microbiology; 41:1152-60

2. Odds FC, 1998: Candida and candidosis; 2nd ed. Balliere Tindall, London, England.

3. Sullivan DJ, Westerneng TJ, Haynes KA, Bennet DE and Coleman DC: Candida dubliniensis sp nov: phenotypic and molecular characterization of novel species associated with oral candidosis in HIV infected individuals; Microbiology: 141:1507-21

WE CLAIM:

1. A method for detecting the presence of at least one Candida spp in a test sample comprising the steps of:

incubating the test sample with at least one peptide with SEQ ID NOs. 1, homolog or a fragment thereof;

detecting the binding of the peptide to the sample in at least one measurable binding assay;

quantitating the intensity of binding of the peptide to the sample; and

comparing the binding and/or intensity with at least one control sample.

2. A method for detecting the presence of at least one Candida spp. in a test sample comprising the steps of:

incubating the test sample with at least one amino acid sequence making up a peptide selected from SEQ ID 1, homologue or a fragment thereof; detecting the binding of the peptide to the sample in at least one measurable binding assay;

quantitating the intensity of binding of the peptide to the sample; and comparing the binding and/or intensity with at least one control sample.

3. A method for detecting the presence of at least one Candida spp. in a test sample comprising the steps of:

incubating the test sample with host cells expressing at least one peptide selected from SEQ ID 1 or a homologue or a fragment thereof; detecting the binding of the peptide to the sample in at least one measurable binding assay;

quantitating the intensity of binding of the peptide to the sample; and comparing the binding and/or intensity with at least one control sample.

4. A method for detecting the presence of at least one Candida spp. in a test sample comprising the steps of:

incubating the test sample with host cells capable of binding to at least one Candida species;

detecting the binding of the host cells to the sample in at least one measurable binding assay;

quantitating the intensity of binding of the peptide to the sample; and

comparing the binding and/or intensity with at least one control sample.

5. The method of any one of the claims 1 to 4, wherein the Candida Spp. is selected from at least one of the species of Candia including C. glabrata, C. dubliniensis, C. guilliermondii, C. parapsilosis, Crytococcus neoformans, Aspergillus sp. C. tropiccdis and C. kefy.

6. The method of any one of the claims 1 to 4, wherein the SEQ ID 1 refers to a generic 12mer peptide capable of binding to Candida spp. cells.

7. The method of claim 5, wherein the SEQ ID 1 has following generic representative sequence:

[E/S/AyT]XX[A/S/T/P/H]X[ILPMA][IVMHPS][PSTDQN][STAPFL][S ATRYL]X, where X is any amino acid.

8. The method of any one of the claims 1 to 4, wherein the measurable binding assay is a cell free assays selected from but not limiting to fluorometric assay or flow cytometric assay.

9. The method of any one of the claims 1 to 4, wherein the detecting or quantitating can be carried out by assays selected from but not limiting to enzyme-linked immunoassay (ELISA or EIA) or immunofluorescence.

10. An apparatus for the detection of Candida spp. comprising an inert means with at least one chamber for holding a test sample and optionally at least one control chamber.

11. The apparatus as claimed in claim 10, wherein the surface of the chamber is pre-coated with at least one of the :

- cells extracted from a culture of Candida spp.;

- an amino acid sequence making up peptide with SEQ ID NO: 1, homologue or a fragment thereof;

- a peptide having SEQ ID NO: 1, homologue or a fragment thereof;

- a host cell expressing at least one amino acid sequence selected from derivative of SEQ ID NO: 1 or a homologue or a fragment thereof or

- a host cell capable of binding to at least one Candida spp.

12. A kit for the detection of Candida spp. in a given samples comprising an apparatus for detection of Candida spp. as claimed in claims 10 - 11; reagent 1 and reagent 2.

13. The kit as claimed in claim 12, wherein, the reagent 1 comprises phage clone or at least one peptide selected from the derivative of SEQ ID NO: 1, homolog or a fragment thereof, conjugated with an enzyme.

14. The kit as claimed in claim 12, wherein, the reagent 2 comprises a substrate for the enzyme of reagent 1.